Most eukaryotic and prokaryotic cells respond to high temperature and certain other stresses with the production of heat shock proteins (HSPs). The HSPs are evolutionarily conserved proteins hypothesized to be essential for survival during stress, or recovery from stress. HSP functions are also known to participate in normal cell growth and development. Very recently, it has been found that specific HSPs are localized to semiautonomous eukaryotic organelles, the mitochondria and chloroplasts. These organelle HSPs are homologous to previously characterized HSPs found in the cytoplasm or nucleus. I have cloned and sequenced the gene for a major, 21 kDa nuclear-encoded chloroplast HSP which is homologous to eukaryotic cytoplasmic low molecular weight HSPs. I have also identified two high molecular weight (63 and 100 kDa) chloroplast HSPs. The proposed research will examine the expression and function of the 21 kDa chloroplast HSP, and further characterize the 63 and 100 kDa chloroplast HSPs. Our long term goals are to determine the function of organelle HSPs at the molecular level, and to better understand their role in the cellular physiology of eukaryotic responses to the environment. Complementary molecular, biochemical and genetic approaches will be used to investigate the role of organelle HSPs. The biochemical properties of HSP21, its interaction with other proteins and intra- organelle distribution will be examined. The expression of HSP21 mRNA and protein will be studied under a range of physiological stress conditions and during normal development. As an additional approach to determining organelle HSP function, mutagenized population of Arabidopsis plants will be screened to identify HSP21 deficient mutants. The phenotype of these mutants will be examined during both normal growth and high temperature stress. Studies of the 63 and 100 kDa HSPs will be initiated, including cloning and sequencing of corresponding cDNAs. In summary, the production of organelle-localized HSPs is a newly discovered component of the stress response and normal developmental program of eukaryotic cells. The proposed studies with further our knowledge of this fundamental aspect of cellular biochemistry.